Vol. 174, No. 24
JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 8057-8064
0021-9193/92/248057-08$02.00/0 Copyright © 1992, American Society for Microbiology
Role of PhoU in Phosphate Transport and Alkaline Phosphatase Regulation M. MUDA, N. N. RAO, AND A. TORRIANI* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 4 August 1992/Accepted 6 October 1992
The negative regulatory function of PhoU in alkaline phosphatase (AP) synthesis was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-(-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in P, transport and in the regulatory role of the phosphate-specific transport system.
To overcome the depletion of free Pi Escherichia coli has evolved a system to scavenge other sources of phosphate from the medium. A number of proteins involved in the transport and metabolism of phosphorylated molecules are induced; one is alkaline phosphatase (AP) (35). A total of 81 proteins have been identified as phosphate regulated (19a). About 20 promoters have been recognized as being phosphate starvation induced (psi promoters). Some of these promoters (17) are regulated by two proteins, PhoB and PhoR, and share a common consensus sequence, the Pho box (15, 16). The PhoR and PhoB proteins are homologous to the family of prokaryotic two-component regulators that are involved in signal transduction (31). The PhoR protein (called the sensor) is a histidine kinase and phosphorylates PhoB (called the regulator), which in turn activates the transcription of the genes with the Pho box. Genetic evidence suggests that PhoR behaves as a protein phosphatase in the presence of high phosphate concentrations, causing the repression of the phosphate regulon (40). Furthermore, genetic evidence indicates that components of the phosphate-specific transport system (Pst) also exert a negative control on AP synthesis (35). This transport system is analogous and homologous to other binding protein-dependent transport systems of various bacteria (11, 22, 27). It consists of a periplasmic Pi binding protein, two transmembrane proteins (PstA and PstC), and an ATP binding protein (PstB). The corresponding genes (pstS, pstA, pstC, and pstB) are part of an operon that includes another gene (phoU). The sequences of these genes are known (2, 3, 34), but only proteins PstS and PhoU have been purified. PhoU is a small protein of 27 kDa (19, 33); its function is unknown. Only one mutation ofphoU (phoU35) is available (36). It is a missense mutation in which alanine 147 is replaced by glutamate (7a). The phenotype caused by this mutation is constitutive synthesis of AP, but the mutation has no effect on the transport of Pi through the Pst (34). By analogy with the regulation of nitrogen utilization, it has been suggested (31) that PhoU may function like PII (24) and may activate the phosphatase function of PhoR. This function has been suggested even though phoU is coregu-
*
lated with all the genes of the Pho regulon and is induced during Pi starvation (34). To overcome this paradox, researchers have hypothesized that PhoU is not functional as a repressor as long as it is in association with the Pst complex during P1 starvation. However, when the Pst complex is reloaded with Pi, PhoU is free in the cytoplasm and shuts off the Pho regulon by activating the phosphatase function of PhoR in the membrane (37). In this paper, we report the construction of a deletion in the phoU gene and its effect on Pi transport and on AP synthesis. Furthermore, the effect of increasing the levels of PhoU on AP induction was analyzed with an approach similar to the one used to analyze the role of several proteins in bacterial chemotaxis (12, 29). MATERUILS AND METHODS Chemicals. Sodium dodecyl sulfate (SDS), acrylamide, molecular weight markers, and all the chemicals used for gel electrophoresis were purchased from Bio-Rad Laboratories (Richmond, Calif.). Isopropyl-3-D-thiogalactopyranoside (IPTG) and deoxynucleotides were obtained from Pharmacia Inc. (Piscataway, N.J.). Agarose used for restriction analysis was obtained from Bethesda Research Laboratories Inc. (Gaithersburg, Md.). Agarose (genetic technology grade) used for DNA fragment purification was obtained from FMC Corporation (Rockland, Maine). Nitrocellulose membranes used for Western blotting (immunoblotting) were obtained from Amersham (Arlington Heights, Ill.). DEAE-cellulose membranes (NA45) used for DNA fragment purification were obtained from Schleicher & Schuell, Inc. (Keene, N.H.). 3-(N-Morpholine)propanesulfonic acid (MOPS) and the antibiotics ampicillin, kanamycin, and chloramphenicol were purchased from Sigma Chemical Co. (St. Louis, Mo.). Restriction endonucleases, T4 DNA ligase, and the Klenow fragment of E. coli DNA Poll were purchased from New England BioLabs, Inc. (Beverly, Mass.), or from Bethesda Research Laboratories. Media. Luria broth supplemented whenever necessary with the appropriate antibiotics (ampicillin, chloramphenicol, and kanamycin at concentrations of 100, 35, and 30 ,ug/ml, respectively) was used. Strain BL21(DE3) used for the overexpression of PhoU was grown in M9ZB medium (32) supplemented with ampicillin at 100 p,g/ml. MOPS-
Corresponding author. 8057
8058
J. BAcTERiOL.
MUDA ET AL.
Plasmids. Plasmid pAN36, which carries the entire pst obtained from Cox et al. (8). A DNA fragment
TABLE 1. Bacterial strains and plasmids
operon, was
Strain or plasmid
Strains HB101 BL21(DE3)
Relevant genotypea
AT854
F- hsd-20 recA13 F- hsdS gal(int::P lacUV5-T7 gene 1 inm21 minS) F- recDl014 K10 pstS25(Am) pit-10 V355 AphoU::Kamr K10 Hfr pit-10 T2r rel tonA recAl srl::TnlO K10 AphoU::Kamr recAl pit-10
AT855 AT857 AT859 AT862 AT870 AT872 AT874
AT854 with pACYC184 K10 recAl pit-JO phoU35 AT854 with pMM7 AT854 with ptacl2 laclq AT857 with pACYC184 AT854 with pDL13 phoU+ AT857 with pDL13 phoU+
V355 C31 MM1 AT833
Plasmids pUC4K pET-3
pACYC184 laclq pAN36
pDL13 pDL17
pMMl
Reference
6 32 7
Our collection This work This work
This work (transduced from MM1) This work This work This work This work This work This work This work
pBR322 derivative carrying Kamr from Tn9O3; Ampr
Pharmacia
pBR322 derivative used for protein expression; Ampr Carries laclq in the EcoRI site of pACYC184; Cmr pACYC184 derivative carrying ginS and the complete pst operon; Cmr pAN36 derivative carrying the complete pst operon; Cmr pAN36 derivative carrying
32
ApstB phoU+; Cmr pET-3 derivative carrying
or
source
M. Malamy 8
This work This work This work
ApstB phoU+ from pDL17;
Ampr pMM2
pMM5
Like pMM1 but carrying ApstB phoU+ in the reverse orientation; Ampr pET-3 derivative carrying pstB+ phoU+ from pDL13;
This work
This work
Ampr
ptacl2 pMM7
pBR322 derivative carrying the tac promoter; Ampr ptacl2 derivative carrying ApstBphoU+ from pMM1 and lacIq from pACYC184 laclq; Ampr
1
This work
I Amp', ampicillin resistance; Cmr, chloramphenicol resistance; Kamr, kanamycin resistance; T2r, resistance to phage T2.
buffered minimal medium (20) was used with glucose as the carbon source and with K2HPO4 as the Pi source. Bacterial strains. The E. coli strains used in this work are described in Table 1. All K10 strains utilized in this study contained a missense mutation in the pit gene coding for a low-efficiency Pi transport system (25) and had an arsenateresistant phenotype. The pst mutations of interest (e.g.,
phoU35 and AphoU)
were
transduced into K10 pit-10 and
selected by linked antibiotic resistance. All K10 derivatives were made recA by P1 transduction and Tetr selection. Strain HB101 was routinely used for plasmid construction. Strain BL21(DE3) was used to overexpress PhoU from
plasmid pMM1.
between the AvaI site in the vector and the AvaI site in the the remainder of the plasmid was allowed to ligate. This plasmid (pDL17) contained a partial deletion of the pstB gene and a complete phoU gene flanked by the terminator of the pst operon (Fig. 1). The ends of the 2.2-kb AvaI-HpaI fragment of pDL17 were blunted by use of the Klenow fragment, and BamHI linkers were added to the fragment. After restriction digestion with BamHI, this fragment was inserted into the unique BamHI site of vector pET-3 to make plasmid pMM1 (Fig. 1). Plasmid pET-3 was kindly provided by W. Studier (Brookhaven National Laboratory, New York, N.Y.). Plasmid ptacl2 was a gift from B. Sauer (Massachusetts Institute of Technology, Cambridge, Mass.). Plasmid pACYC184 laclq carrying the lacIl gene in the EcoRI site of pACYC184 was from M. Malamy (Tufts University, Boston, Mass.). Plasmid pUC4K was from Pharmacia. Plasmid DNA was prepared as described by Sambrook et al. (28). Restriction fragments were purified from agarose (genetic technology grade) by blotting on DEAE-cellulose membranes (NA45) in accordance with the instruction of the manufacturer. Plasmids were introduced into different strains as described by Ausubel et al. (4). Plasmid pMM5 (Fig. 1) was constructed from plasmid pDL13 (Fig. 1) by subcloning of the HpaI-HpaI fragment carryingpstB and phoU into pET-3. In plasmid pMM7 (Fig. 1), the phoU gene was inserted downstream from the tac promoter of plasmid ptacl2 (1). The unique PvuII site of plasmid ptacl2 was converted to BamHI by the addition of nucleotide linkers. Subsequently, the 2.3-kb fragment obtained by digestion of pMM5 with BamHI was ligated in the newly created BamHI site. Regulated expression of phoU was obtained by ligating the 1.3-kb EcoRI fragment, which carries the laclIq gene from plasmid pACYC184 lacli, into the unique EcoRI site of ptacl2 (Fig. 1). Cell extraction and antibody preparation. Strain BL21(DE3) carrying plasmid pMM1 was grown in M9ZB medium. At an optical density at 540 nm (OD540) of 0.6, the cells were induced by the addition 0.4 mM IPTG, harvested after 3 h by centrifugation at 8,000 x g for 10 min, resuspended in 50 mM Tris-HCl buffer (pH 8) with 1 mM EDTA, and lysed with lysozyme (100 ,g/ml). After 30 min at 4°C, the extract was sonicated three times for 30 s each time with a Branson Sonifier at a 45-W output. The extract was centrifuged at 15,000 x g for 15 min. The pellet was dissolved in SDS loading buffer (50 mM Tris-HCl [pH 6.8] with 100 niM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), and the solution was heated at 95°C for 5 min. The proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) (see Fig. 2). The PhoU protein (27 kDa) was detected with 1 M KCl (5). The gel slice containing the PhoU protein was minced in 0.1x running buffer (lx running buffer is 25 mM Tris base, 250 mM glycine (pH 8.3), and only 0.1% SDS) and electroeluted with a Little Blue Tank apparatus (ISCO). About 200 ,ug of protein was emulsified with complete Freund's adjuvant and injected into a New Zealand White male rabbit. Subsequently, two boosts of 100 ,ug of protein PhoU each were administered at 2-week intervals. Western blot analysis of PhoU. PAGE of proteins was done with a Miniprotean gel system (Bio-Rad) by the method of Laemmli (13). Electroblotting of proteins on nitrocellulose membranes for immunodetection was performed as described previously (28). The antiserum was diluted 1:1,000 in pst region was deleted by digestion withAvaI;
VOL. 174, 1992
ROLE OF PhoU IN PHOSPHATE TRANSPORT AND AP REGULATION 4*
~~X
._
I
0
10.75 kb
*0
4.2 5 kb
-
8059
._
I
CD
I
L
I
11
pstS pstC pstA pstB
I
phoU
pAN36 15 kb
BamHI-Bgill 10.4 kb fragment Ugate
Xholl (BamHI/BgIll) coRI BamHI / BgIll
pET-3
BamHI
ptacphoU Intermediate 4.8kb
BamHI
phoU FIG. 1. Construction of plasmids.
5% (wt/vol) skim milk in phosphate-buffered saline, and the secondary antibodies, conjugated with horseradish peroxidase (Bio-Rad), were also diluted 1:1,000. The developing reaction was carried out as described previously (4). AP activity. AP activity was measured as previously described (14). One unit of enzyme activity corresponded to the hydrolysis of 1 nmol of p-nitrophenyl phosphate per min and per 2 x 108 cells per ml (OD540, 1.0; 0.38 mg [dry weight] of bacteria). Construction of a phoU chromosomal deletion. A partial deletion of phoU was constructed by the method of Shevell et al. (30). DNA from plasmid pMM5 was digested with NcoI
and MluI. After the creation of blunt ends, the larger fragment was ligated to the 1.3-kb HincIl fragment from plasmid pUC4K (Pharmacia), which carries a kanamycin resistance cassette. This construct contained a deletion of 347 bp internal to the phoU gene, leaving only 26 amino acids of the protein at the N terminus. To avoid interference in the expression of the nearby pst genes, we kept the promoter of the kanamycin resistance cassette in the same orientation as the genes in the pst operon. The plasmid so constructed (pMM5 Kam') was linearized by digestion with EcoRI and SalI. About 2 p.g of the 3.6-kb fragment carrying the deletion was used to transform strain V355.
8060
J. BAcTERIOL.
MUDA ET AL.
We obtained one transformant that was AP constitutive, Kamr, and Amps. This strain, V355 AphoU::Kamr (MM1), was used as the donor to transduce, via P1 (18), the deletion into other strains. The proper construction of the deletion was confirmed by Western blotting to detect the absence of the PhoU protein in Pi-rich and Pi-starved cells and by Southern hybridization. Southern hybridization analysis of the phoU deletion. Chromosomal DNAs prepared from E. coli K10 and from E. coli AphoU strains were digested with HpaI and HincIl. Plasmid pMM7 DNA was digested with BamHI. These restricted DNA preparations were then separated on a 1.0% agarose gel with TBE buffer (28). The chromosomal and plasmid pMM5 DNA fragments from the agarose gel were transferred to a nitrocellulose filter (Schleicher & Schuell BA85) as described previously (28). The probe was prepared from plasmid pMM5. This plasmid was restricted with BamHI, and the 2.3-kb fragment containing pstB and phoU was isolated by use of a DEAE-cellulose membrane (Schleicher & Schuell NA45). The probe was labelled with [cx-32P]dATP by nick translation (28). Hybridization was done at 65°C. Autoradiograms were prepared with XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) by exposing the membrane for 1 day at -70°C before development. Amino acid sequencing of PhoU produced by pMM7. Sequencing of the first 10 N-terminal amino acids of PhoU produced by plasmid pMM7 was done by automated amino acid sequencing (using the protein sequencing facilities at the Whitehead Institute, Cambridge, Mass.). PhoU was extracted from cells of strain AT859 carrying plasmid pMM7 and grown in MOPS-buffered medium with 2 mM Pi and 1 mM IPTG. The cells were suspended in SDS-Tris-HCl gel buffer, and the suspension was boiled for 5 min and fractionated by SDS-PAGE. From the gel, the proteins were transferred to an Immobilon-P transfer membrane (Millipore, Bedford, Mass.). The 27-kDa band was used for sequencing. P1 transport. K10 pit-10 cells grown for five to seven generations overnight in MOPS-glucose (2 mg/ml)-Pi (2 mM) medium were diluted 12.5-fold in the same medium. After two generations (OD540, ca. 0.50), they were centrifuged at 10,000 x g for 10 min at room temperature (20°C), washed twice with MOPS medium free of Pi and glucose, and resuspended to an OD540 of ca. 0.5 in MOPS medium free of Pi but rich in glucose (6 mg/ml). Pi starvation was accomplished by shaking the culture at 37°C. The duration of starvation was dictated by the increase in cell mass and AP activity. After a 20% increase, growth stopped and AP activity reached a plateau; the cells were considered starved. Uptake was started by adding 0.5 mM 32pO4 (0.1 ,Ci/ml) to the starved cells. This concentration is saturating, since the Km of transport of the Pst is 0.2 ,uM, as observed by Willsky and Malamy (38, 39). Samples of 50 ,ul were withdrawn every 15 s, rapidly filtered through Millipore membranes, and washed with 10 ml of 0.1 M Tris-HCl (pH 7.4). The Vma, of Pi transport was calculated as micromoles of Pi per minute per milligram (dry weight) from the initial rate at 15 to 30 s. To measure the K, of Pi transport, we added increasing amounts of K2HPO4 with 0.1 ,uCi of 32pO4 per ml to the P1-starved cells. AP activity was measured before and after Pi starvation.
RESULTS Analysis of the PhoU protein. Massive overproduction of a 27-kDa protein was observed (Fig. 2, lane 3) in BL21(DE3)derived strains with the plasmid carrying the phoU+ gene
.'L.. -M-4,.
97.4
.i
66.2 45
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f..- t -jm,z,z;z-* -'Am"AMM.
'IWI"
31
0
40"01
21 5 *-
l14 .:
".
'Aki
5 6 3 4 2 FIG. 2. Analysis by SDS-PAGE of PhoU overproduced by strain BL21(DE3) carrying plasmid pMM1 phoU+ (lanes 3, 5, and 6) or pMM2phoU+ (lanes 2 and 4) in the reverse orientation. Lanes: 1, molecular weight standards (in thousands); 2 and 3, crude extracts; 4 and 5, pellets obtained after centrifugation at 15,000 x g; 6, supernatant. Protein PhoU is the band at 27 kDa. Each lane was loaded with the same amount of sample, corresponding to about 108 cells. Electrophoresis was done on a 12% polyacrylamide gel, and the samples were prepared as described in the text.
under the control of the T7 promoter (pMM1). In the fractionated extract (centrifuged at 15,000 x g), most of the PhoU protein was in the pellet (Fig. 2, lane 5), a result that suggested that PhoU was aggregated into inclusion bodies. A similar result was previously reported by Surin et al. (34). However, some of the PhoU protein was also observed in the supernatant (Fig. 2, lane 6). The strain carrying plasmid pMM2, which bears the insert of PhoU in the reverse orientation, was used as a control (Fig. 2, lanes 2 and 4). The purified PhoU protein was used to immunize a rabbit (see Materials and Methods). The serum produced yielded a clear immunological reaction with a 27-kDa band representing a sample from an extract of wild-type K10 grown under conditions of Pi starvation (Fig. 3, lane 5). It is worth noting that the protein was also present in cells grown with excess Pi (Fig. 3, lane 6). This serum also reacted with a protein produced by the phoU35 mutant (Fig. 3, lanes 3 and 4), suggesting that the mutant protein was stable and that the mutation was a missense mutation, in agreement with the DNA sequence obtained by Cox (7a). Effect of a phoU chromosomal deletion. A phoU deletion
27K
1 2 3 4 56 FIG. 3. Immunoblot of the PhoU protein. Cells grown in MOPSglucose medium containing 0.1 mM K2HP04 were harvested by centrifugation at the mid-log phase or at the beginning of the stationary phase and resuspended in loading buffer as described in the text. The same amount of protein, corresponding to -iO' cells, was loaded in each lane. Lanes: 1 and 2, strain AT854 AphoU in the stationary and exponential phases of growth, respectively; 3 and 4, strain C4 phoU3S in the stationary and exponential phases of growth, respectively; 5 and 6, wild-type strain K10 in the stationary and exponential phases of growth, respectively.
VOL. 174, 1992
ROLE OF PhoU IN PHOSPHATE TRANSPORT AND AP REGULATION I
8061
+
-°0a
0
1-c
JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 8057-8064
0021-9193/92/248057-08$02.00/0 Copyright © 1992, American Society for Microbiology
Role of PhoU in Phosphate Transport and Alkaline Phosphatase Regulation M. MUDA, N. N. RAO, AND A. TORRIANI* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 4 August 1992/Accepted 6 October 1992
The negative regulatory function of PhoU in alkaline phosphatase (AP) synthesis was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-(-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in P, transport and in the regulatory role of the phosphate-specific transport system.
To overcome the depletion of free Pi Escherichia coli has evolved a system to scavenge other sources of phosphate from the medium. A number of proteins involved in the transport and metabolism of phosphorylated molecules are induced; one is alkaline phosphatase (AP) (35). A total of 81 proteins have been identified as phosphate regulated (19a). About 20 promoters have been recognized as being phosphate starvation induced (psi promoters). Some of these promoters (17) are regulated by two proteins, PhoB and PhoR, and share a common consensus sequence, the Pho box (15, 16). The PhoR and PhoB proteins are homologous to the family of prokaryotic two-component regulators that are involved in signal transduction (31). The PhoR protein (called the sensor) is a histidine kinase and phosphorylates PhoB (called the regulator), which in turn activates the transcription of the genes with the Pho box. Genetic evidence suggests that PhoR behaves as a protein phosphatase in the presence of high phosphate concentrations, causing the repression of the phosphate regulon (40). Furthermore, genetic evidence indicates that components of the phosphate-specific transport system (Pst) also exert a negative control on AP synthesis (35). This transport system is analogous and homologous to other binding protein-dependent transport systems of various bacteria (11, 22, 27). It consists of a periplasmic Pi binding protein, two transmembrane proteins (PstA and PstC), and an ATP binding protein (PstB). The corresponding genes (pstS, pstA, pstC, and pstB) are part of an operon that includes another gene (phoU). The sequences of these genes are known (2, 3, 34), but only proteins PstS and PhoU have been purified. PhoU is a small protein of 27 kDa (19, 33); its function is unknown. Only one mutation ofphoU (phoU35) is available (36). It is a missense mutation in which alanine 147 is replaced by glutamate (7a). The phenotype caused by this mutation is constitutive synthesis of AP, but the mutation has no effect on the transport of Pi through the Pst (34). By analogy with the regulation of nitrogen utilization, it has been suggested (31) that PhoU may function like PII (24) and may activate the phosphatase function of PhoR. This function has been suggested even though phoU is coregu-
*
lated with all the genes of the Pho regulon and is induced during Pi starvation (34). To overcome this paradox, researchers have hypothesized that PhoU is not functional as a repressor as long as it is in association with the Pst complex during P1 starvation. However, when the Pst complex is reloaded with Pi, PhoU is free in the cytoplasm and shuts off the Pho regulon by activating the phosphatase function of PhoR in the membrane (37). In this paper, we report the construction of a deletion in the phoU gene and its effect on Pi transport and on AP synthesis. Furthermore, the effect of increasing the levels of PhoU on AP induction was analyzed with an approach similar to the one used to analyze the role of several proteins in bacterial chemotaxis (12, 29). MATERUILS AND METHODS Chemicals. Sodium dodecyl sulfate (SDS), acrylamide, molecular weight markers, and all the chemicals used for gel electrophoresis were purchased from Bio-Rad Laboratories (Richmond, Calif.). Isopropyl-3-D-thiogalactopyranoside (IPTG) and deoxynucleotides were obtained from Pharmacia Inc. (Piscataway, N.J.). Agarose used for restriction analysis was obtained from Bethesda Research Laboratories Inc. (Gaithersburg, Md.). Agarose (genetic technology grade) used for DNA fragment purification was obtained from FMC Corporation (Rockland, Maine). Nitrocellulose membranes used for Western blotting (immunoblotting) were obtained from Amersham (Arlington Heights, Ill.). DEAE-cellulose membranes (NA45) used for DNA fragment purification were obtained from Schleicher & Schuell, Inc. (Keene, N.H.). 3-(N-Morpholine)propanesulfonic acid (MOPS) and the antibiotics ampicillin, kanamycin, and chloramphenicol were purchased from Sigma Chemical Co. (St. Louis, Mo.). Restriction endonucleases, T4 DNA ligase, and the Klenow fragment of E. coli DNA Poll were purchased from New England BioLabs, Inc. (Beverly, Mass.), or from Bethesda Research Laboratories. Media. Luria broth supplemented whenever necessary with the appropriate antibiotics (ampicillin, chloramphenicol, and kanamycin at concentrations of 100, 35, and 30 ,ug/ml, respectively) was used. Strain BL21(DE3) used for the overexpression of PhoU was grown in M9ZB medium (32) supplemented with ampicillin at 100 p,g/ml. MOPS-
Corresponding author. 8057
8058
J. BAcTERiOL.
MUDA ET AL.
Plasmids. Plasmid pAN36, which carries the entire pst obtained from Cox et al. (8). A DNA fragment
TABLE 1. Bacterial strains and plasmids
operon, was
Strain or plasmid
Strains HB101 BL21(DE3)
Relevant genotypea
AT854
F- hsd-20 recA13 F- hsdS gal(int::P lacUV5-T7 gene 1 inm21 minS) F- recDl014 K10 pstS25(Am) pit-10 V355 AphoU::Kamr K10 Hfr pit-10 T2r rel tonA recAl srl::TnlO K10 AphoU::Kamr recAl pit-10
AT855 AT857 AT859 AT862 AT870 AT872 AT874
AT854 with pACYC184 K10 recAl pit-JO phoU35 AT854 with pMM7 AT854 with ptacl2 laclq AT857 with pACYC184 AT854 with pDL13 phoU+ AT857 with pDL13 phoU+
V355 C31 MM1 AT833
Plasmids pUC4K pET-3
pACYC184 laclq pAN36
pDL13 pDL17
pMMl
Reference
6 32 7
Our collection This work This work
This work (transduced from MM1) This work This work This work This work This work This work This work
pBR322 derivative carrying Kamr from Tn9O3; Ampr
Pharmacia
pBR322 derivative used for protein expression; Ampr Carries laclq in the EcoRI site of pACYC184; Cmr pACYC184 derivative carrying ginS and the complete pst operon; Cmr pAN36 derivative carrying the complete pst operon; Cmr pAN36 derivative carrying
32
ApstB phoU+; Cmr pET-3 derivative carrying
or
source
M. Malamy 8
This work This work This work
ApstB phoU+ from pDL17;
Ampr pMM2
pMM5
Like pMM1 but carrying ApstB phoU+ in the reverse orientation; Ampr pET-3 derivative carrying pstB+ phoU+ from pDL13;
This work
This work
Ampr
ptacl2 pMM7
pBR322 derivative carrying the tac promoter; Ampr ptacl2 derivative carrying ApstBphoU+ from pMM1 and lacIq from pACYC184 laclq; Ampr
1
This work
I Amp', ampicillin resistance; Cmr, chloramphenicol resistance; Kamr, kanamycin resistance; T2r, resistance to phage T2.
buffered minimal medium (20) was used with glucose as the carbon source and with K2HPO4 as the Pi source. Bacterial strains. The E. coli strains used in this work are described in Table 1. All K10 strains utilized in this study contained a missense mutation in the pit gene coding for a low-efficiency Pi transport system (25) and had an arsenateresistant phenotype. The pst mutations of interest (e.g.,
phoU35 and AphoU)
were
transduced into K10 pit-10 and
selected by linked antibiotic resistance. All K10 derivatives were made recA by P1 transduction and Tetr selection. Strain HB101 was routinely used for plasmid construction. Strain BL21(DE3) was used to overexpress PhoU from
plasmid pMM1.
between the AvaI site in the vector and the AvaI site in the the remainder of the plasmid was allowed to ligate. This plasmid (pDL17) contained a partial deletion of the pstB gene and a complete phoU gene flanked by the terminator of the pst operon (Fig. 1). The ends of the 2.2-kb AvaI-HpaI fragment of pDL17 were blunted by use of the Klenow fragment, and BamHI linkers were added to the fragment. After restriction digestion with BamHI, this fragment was inserted into the unique BamHI site of vector pET-3 to make plasmid pMM1 (Fig. 1). Plasmid pET-3 was kindly provided by W. Studier (Brookhaven National Laboratory, New York, N.Y.). Plasmid ptacl2 was a gift from B. Sauer (Massachusetts Institute of Technology, Cambridge, Mass.). Plasmid pACYC184 laclq carrying the lacIl gene in the EcoRI site of pACYC184 was from M. Malamy (Tufts University, Boston, Mass.). Plasmid pUC4K was from Pharmacia. Plasmid DNA was prepared as described by Sambrook et al. (28). Restriction fragments were purified from agarose (genetic technology grade) by blotting on DEAE-cellulose membranes (NA45) in accordance with the instruction of the manufacturer. Plasmids were introduced into different strains as described by Ausubel et al. (4). Plasmid pMM5 (Fig. 1) was constructed from plasmid pDL13 (Fig. 1) by subcloning of the HpaI-HpaI fragment carryingpstB and phoU into pET-3. In plasmid pMM7 (Fig. 1), the phoU gene was inserted downstream from the tac promoter of plasmid ptacl2 (1). The unique PvuII site of plasmid ptacl2 was converted to BamHI by the addition of nucleotide linkers. Subsequently, the 2.3-kb fragment obtained by digestion of pMM5 with BamHI was ligated in the newly created BamHI site. Regulated expression of phoU was obtained by ligating the 1.3-kb EcoRI fragment, which carries the laclIq gene from plasmid pACYC184 lacli, into the unique EcoRI site of ptacl2 (Fig. 1). Cell extraction and antibody preparation. Strain BL21(DE3) carrying plasmid pMM1 was grown in M9ZB medium. At an optical density at 540 nm (OD540) of 0.6, the cells were induced by the addition 0.4 mM IPTG, harvested after 3 h by centrifugation at 8,000 x g for 10 min, resuspended in 50 mM Tris-HCl buffer (pH 8) with 1 mM EDTA, and lysed with lysozyme (100 ,g/ml). After 30 min at 4°C, the extract was sonicated three times for 30 s each time with a Branson Sonifier at a 45-W output. The extract was centrifuged at 15,000 x g for 15 min. The pellet was dissolved in SDS loading buffer (50 mM Tris-HCl [pH 6.8] with 100 niM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), and the solution was heated at 95°C for 5 min. The proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) (see Fig. 2). The PhoU protein (27 kDa) was detected with 1 M KCl (5). The gel slice containing the PhoU protein was minced in 0.1x running buffer (lx running buffer is 25 mM Tris base, 250 mM glycine (pH 8.3), and only 0.1% SDS) and electroeluted with a Little Blue Tank apparatus (ISCO). About 200 ,ug of protein was emulsified with complete Freund's adjuvant and injected into a New Zealand White male rabbit. Subsequently, two boosts of 100 ,ug of protein PhoU each were administered at 2-week intervals. Western blot analysis of PhoU. PAGE of proteins was done with a Miniprotean gel system (Bio-Rad) by the method of Laemmli (13). Electroblotting of proteins on nitrocellulose membranes for immunodetection was performed as described previously (28). The antiserum was diluted 1:1,000 in pst region was deleted by digestion withAvaI;
VOL. 174, 1992
ROLE OF PhoU IN PHOSPHATE TRANSPORT AND AP REGULATION 4*
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pET-3
BamHI
ptacphoU Intermediate 4.8kb
BamHI
phoU FIG. 1. Construction of plasmids.
5% (wt/vol) skim milk in phosphate-buffered saline, and the secondary antibodies, conjugated with horseradish peroxidase (Bio-Rad), were also diluted 1:1,000. The developing reaction was carried out as described previously (4). AP activity. AP activity was measured as previously described (14). One unit of enzyme activity corresponded to the hydrolysis of 1 nmol of p-nitrophenyl phosphate per min and per 2 x 108 cells per ml (OD540, 1.0; 0.38 mg [dry weight] of bacteria). Construction of a phoU chromosomal deletion. A partial deletion of phoU was constructed by the method of Shevell et al. (30). DNA from plasmid pMM5 was digested with NcoI
and MluI. After the creation of blunt ends, the larger fragment was ligated to the 1.3-kb HincIl fragment from plasmid pUC4K (Pharmacia), which carries a kanamycin resistance cassette. This construct contained a deletion of 347 bp internal to the phoU gene, leaving only 26 amino acids of the protein at the N terminus. To avoid interference in the expression of the nearby pst genes, we kept the promoter of the kanamycin resistance cassette in the same orientation as the genes in the pst operon. The plasmid so constructed (pMM5 Kam') was linearized by digestion with EcoRI and SalI. About 2 p.g of the 3.6-kb fragment carrying the deletion was used to transform strain V355.
8060
J. BAcTERIOL.
MUDA ET AL.
We obtained one transformant that was AP constitutive, Kamr, and Amps. This strain, V355 AphoU::Kamr (MM1), was used as the donor to transduce, via P1 (18), the deletion into other strains. The proper construction of the deletion was confirmed by Western blotting to detect the absence of the PhoU protein in Pi-rich and Pi-starved cells and by Southern hybridization. Southern hybridization analysis of the phoU deletion. Chromosomal DNAs prepared from E. coli K10 and from E. coli AphoU strains were digested with HpaI and HincIl. Plasmid pMM7 DNA was digested with BamHI. These restricted DNA preparations were then separated on a 1.0% agarose gel with TBE buffer (28). The chromosomal and plasmid pMM5 DNA fragments from the agarose gel were transferred to a nitrocellulose filter (Schleicher & Schuell BA85) as described previously (28). The probe was prepared from plasmid pMM5. This plasmid was restricted with BamHI, and the 2.3-kb fragment containing pstB and phoU was isolated by use of a DEAE-cellulose membrane (Schleicher & Schuell NA45). The probe was labelled with [cx-32P]dATP by nick translation (28). Hybridization was done at 65°C. Autoradiograms were prepared with XAR-5 film (Eastman Kodak Co., Rochester, N.Y.) by exposing the membrane for 1 day at -70°C before development. Amino acid sequencing of PhoU produced by pMM7. Sequencing of the first 10 N-terminal amino acids of PhoU produced by plasmid pMM7 was done by automated amino acid sequencing (using the protein sequencing facilities at the Whitehead Institute, Cambridge, Mass.). PhoU was extracted from cells of strain AT859 carrying plasmid pMM7 and grown in MOPS-buffered medium with 2 mM Pi and 1 mM IPTG. The cells were suspended in SDS-Tris-HCl gel buffer, and the suspension was boiled for 5 min and fractionated by SDS-PAGE. From the gel, the proteins were transferred to an Immobilon-P transfer membrane (Millipore, Bedford, Mass.). The 27-kDa band was used for sequencing. P1 transport. K10 pit-10 cells grown for five to seven generations overnight in MOPS-glucose (2 mg/ml)-Pi (2 mM) medium were diluted 12.5-fold in the same medium. After two generations (OD540, ca. 0.50), they were centrifuged at 10,000 x g for 10 min at room temperature (20°C), washed twice with MOPS medium free of Pi and glucose, and resuspended to an OD540 of ca. 0.5 in MOPS medium free of Pi but rich in glucose (6 mg/ml). Pi starvation was accomplished by shaking the culture at 37°C. The duration of starvation was dictated by the increase in cell mass and AP activity. After a 20% increase, growth stopped and AP activity reached a plateau; the cells were considered starved. Uptake was started by adding 0.5 mM 32pO4 (0.1 ,Ci/ml) to the starved cells. This concentration is saturating, since the Km of transport of the Pst is 0.2 ,uM, as observed by Willsky and Malamy (38, 39). Samples of 50 ,ul were withdrawn every 15 s, rapidly filtered through Millipore membranes, and washed with 10 ml of 0.1 M Tris-HCl (pH 7.4). The Vma, of Pi transport was calculated as micromoles of Pi per minute per milligram (dry weight) from the initial rate at 15 to 30 s. To measure the K, of Pi transport, we added increasing amounts of K2HPO4 with 0.1 ,uCi of 32pO4 per ml to the P1-starved cells. AP activity was measured before and after Pi starvation.
RESULTS Analysis of the PhoU protein. Massive overproduction of a 27-kDa protein was observed (Fig. 2, lane 3) in BL21(DE3)derived strains with the plasmid carrying the phoU+ gene
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5 6 3 4 2 FIG. 2. Analysis by SDS-PAGE of PhoU overproduced by strain BL21(DE3) carrying plasmid pMM1 phoU+ (lanes 3, 5, and 6) or pMM2phoU+ (lanes 2 and 4) in the reverse orientation. Lanes: 1, molecular weight standards (in thousands); 2 and 3, crude extracts; 4 and 5, pellets obtained after centrifugation at 15,000 x g; 6, supernatant. Protein PhoU is the band at 27 kDa. Each lane was loaded with the same amount of sample, corresponding to about 108 cells. Electrophoresis was done on a 12% polyacrylamide gel, and the samples were prepared as described in the text.
under the control of the T7 promoter (pMM1). In the fractionated extract (centrifuged at 15,000 x g), most of the PhoU protein was in the pellet (Fig. 2, lane 5), a result that suggested that PhoU was aggregated into inclusion bodies. A similar result was previously reported by Surin et al. (34). However, some of the PhoU protein was also observed in the supernatant (Fig. 2, lane 6). The strain carrying plasmid pMM2, which bears the insert of PhoU in the reverse orientation, was used as a control (Fig. 2, lanes 2 and 4). The purified PhoU protein was used to immunize a rabbit (see Materials and Methods). The serum produced yielded a clear immunological reaction with a 27-kDa band representing a sample from an extract of wild-type K10 grown under conditions of Pi starvation (Fig. 3, lane 5). It is worth noting that the protein was also present in cells grown with excess Pi (Fig. 3, lane 6). This serum also reacted with a protein produced by the phoU35 mutant (Fig. 3, lanes 3 and 4), suggesting that the mutant protein was stable and that the mutation was a missense mutation, in agreement with the DNA sequence obtained by Cox (7a). Effect of a phoU chromosomal deletion. A phoU deletion
27K
1 2 3 4 56 FIG. 3. Immunoblot of the PhoU protein. Cells grown in MOPSglucose medium containing 0.1 mM K2HP04 were harvested by centrifugation at the mid-log phase or at the beginning of the stationary phase and resuspended in loading buffer as described in the text. The same amount of protein, corresponding to -iO' cells, was loaded in each lane. Lanes: 1 and 2, strain AT854 AphoU in the stationary and exponential phases of growth, respectively; 3 and 4, strain C4 phoU3S in the stationary and exponential phases of growth, respectively; 5 and 6, wild-type strain K10 in the stationary and exponential phases of growth, respectively.
VOL. 174, 1992
ROLE OF PhoU IN PHOSPHATE TRANSPORT AND AP REGULATION I
8061
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